This project focuses on the characterization and cellular regulation of Ca 2+-activated K+ channels (Kca) in erythrocytes from humans and mice. Cell dehydration of hemoglobin S-containing erythrocytes favors the formation of sickle cells due to increased hemoglobin S polymerization. Our laboratory has shown that cell sickling, in vitro, can be prevented by using clotrimazole, a known antifungal agent. This laboratory has also shown that oral administration of clotrimazole in normal volunteers inhibits the erythrocyte Kca in vivo. These findings suggest the possible use of clotrimazole to prevent cell dehydration and strongly suggest that these channels are important in the formation of sickle cells in vivo. The similarities between K+ homeostasis and erythrocyte dehydration in mouse and human sickle cells reinforces the use of animal models to study possible mechanism that can alter K+ content and cellular hydration status. Hence, our major interest in this project is to provide a thorough understanding of this channel's activity that will contribute to future therapeutic approaches to treat sickle cell anemia in a more specific manner. To this end, we will evaluate the activity of this transport system in mouse and human erythrocytes with normal and S hemoglobin. We have defined three specific objectives to address these issues: 1) Characterize the Kca activity in CD1 mouse erythrocytes. We will measure unidirectional and charybdotoxin sensitive fluxes using 86-Rb to identify possible differences in the kinetics for activation of the Kca caused by novel channel openers like endothelin, guanabenz and interleukin-8 and 10. 2) Test whether Kca of CD1 mouse erythrocytes can be regulated by protein kinase C inhibitors or phosphatase inhibitors. We will study the effect of protein kinase C and phosphatase inhibitors on the opener-induced charybdotoxin- sensitive K+ fluxes in mouse erythrocyte. 3) Test whether human sickle erythrocytes can be activated by the Kca openers. In addition, we will study the regulation of the Kca by phosphorylation and dephosphorylation events. Our long-term objective is to thoroughly characterize potassium transport pathways because in principle these can contribute to cellular volume and hemoglobin S polymerization and to identify specific agents that can prevent cell sickling in sickle cell anemia patients.